Method for treating respiratory distress syndrome

ABSTRACT

The invention provides a method for treating infants, children or adults suffering from pulmonary distress caused by low or insufficient production of surfactant. It is particularly suitable for treating premature infants suffering from Respiratory Distress Syndrome. The method comprises administering a leptin compound to an individual with impaired surfactant production for a time and in an amount sufficient to enhance surfactant production. The method may be used for treatment of any mammal with impaired lung surfactant production.

BACKGROUND

I. Field of Invention

The present invention provides a new method for treating pulmonarydistress caused by low or insufficient surfactant production in infants,children or adults. More particularly, the method of the inventionutilizes leptin to treat individuals with impaired lung surfactantproduction and is particularly useful for treating Respiratory DistressSyndrome (RDS) in premature infants.

II. Background of Invention

Premature infants are at increased risk for developing RespiratoryDistress Syndrome (RDS), the leading cause of neonatal morbidity andmortality in developed countries (Mendelson et al., Bailliere's Clin.Endocrinol. Metab. 4:351-78, 1990). This condition is caused by adeficiency of lung surfactant, a complex material consisting ofphospholipids, neutral lipids, carbohydrate and proteins. These infantsrequire assisted ventilation and supplemental oxygen for prolongedperiods of time. Often these infants develop Bronchopulmonary Dysplasia(BPD), a chronic lung disease associated with neurodevelopmental delay,poor growth, and late mortality (Bader et al., J. Pediatr. 110(5):693-9,1987; Gibson et al., Am. J. Dis. Child. 143(7):721-5, 1988; Kurzner etal., Pediatrics 81(3):379-84, 1988; Vohr et al., Dev. Med. Child.Neurol. 33(8):690-7, 1991). Inflammation, primarily due tooxygen-induced free radical formation, positive pressure ventilation,and infection, is thought to be a key factor in the lung injury observedin these infants (Pierce and Bancalari, Pediatr. Pulmonol. 19(6):371-8,1995).

Strategies aimed at treating the pulmonary inflammation in BPD throughthe use of systemic steroids have not shown a favorable outcome indecreasing the overall incidence of this disease. Three largemulticenter trials, which enrolled a total of 1348 infants,independently showed no significant benefit to early administration(within 72 hours of life) of steroids on the incidence of BPD (Hallidayet al., In: Hot Topics in Neonatology, Ross Laboratories, pp. 267-75,1999; Soll et al., Pediatr. Res. 45: 226A, 1999; Stark et al.,Pediatrics Supplement 104: 739A, 1999). Two of these trials were stoppedprematurely due to concerns of significant detrimental side effects,including gastrointestinal perforation, periventricular leukomalacia,poor weight gain, gastrointestinal hemorrhage, and hypertension. Longterm follow-up studies have shown a significant detrimental effect onsomatic growth (Gibson et al., Arch. Dis. Child 69: 505-9, 1993, Yeh etal., Pediatrics 101(5):E7, 1998; O'Shea et al., Pediatrics 104(1 part1):15-21, 1999). These adverse effects may be related to the cataboliceffects of steroids on growing tissues (Tsai et al., Act. Paediatr.85(12):1487-90, 1996). Efforts to reduce the incidence of BPD usingother strategies such as inhaled steroids, high-frequency ventilation,and treatment of RDS with surfactant have also shown mixed results.There has been some success in reducing the incidence of RDS byenhancing surfactant production in utero via glucocorticoidadministration to mothers in preterm labor (Liggins and Howie,Pediatrics 59:515-25, 1972). However, this treatment strategy isdependent on accurate identification of those mothers at risk forpreterm delivery, and thus, is only effective for a small subset ofpremature infants affected with RDS. Furthermore, despite significantadvances in neonatal care during the past three decades, the incidenceof RDS and BPD has not changed significantly. There remains a clear needto identify alternative treatment strategies for this disease.

As more fully described below, the present invention overcomes theproblems associated with previous forms of RDS therapy and includes anovel method of treating RDS that can be administered to prematureinfants as well as infants, children or adult subjects who havedeficient lung surfactant.

SUMMARY OF THE INVENTION

The present invention includes a method for treating respiratorydistress by treatment with leptin. According to the invention, leptinmay be administered orally as well as by intravenously, intramuscularly,and other parenteral and enteral means. In one embodiment, the inventionincludes a method for treating RDS and BPD in premature infants. Anotheraspect of this invention is its usefulness for treating infants,children or adults suffering from pulmonary distress caused by low orinsufficient production of surfactant. The present invention overcomesthe problems associated with previous forms of RDS therapy, particularlythe use of steroids.

The method of the invention provides for improving lung surfactantproduction in an individual with impaired surfactant production byadministering a leptin compound to the individual for a time and in anamount sufficient to enhance surfactant production. The individual maybe any mammal. Further, while the invention may be used for thetreatment of any individual with impaired lung surfactant production, itis particularly useful for treating infants with intrauterinedevelopment of less than nine months. The leptin compound may compriseat least a biologically active fragment of leptin that is capable ofbinding to the leptin receptor and eliciting a biological effect such asincreased surfactant production, and may be derived from any source ofleptin or a biologically active fragment of leptin including recombinantprotein.

In the method of the invention, the leptin compound is administered in adosage from about 0.1 ng/kg body weight to about 100 mg/kg body weightand by a method selected from the group consisting of subcutaneously,intradermally, intravenously, intramuscularly, intraperitoneally,transdermally, orally, enteral tube feeding, pulmonary delivery,intranasal delivery, controlled release delivery and pump delivery.

Leptin may be administered with nutritional supplements, growth factors,and steroids, such as dexamethasone, that increase lung function. In apreferred embodiment, the growth factors may be selected from the groupconsisting of epidermal growth factor, fibroblast growth factor,insulin-like growth factor, thyroid hormone, and platelet derived growthfactor.

Further, the method of the invention which comprises administering aleptin compound to an individual with impaired lung surfactantproduction for a time and in an amount sufficient to enhance surfactantproduction is particularly suitable for the treatment of individualswith Respiratory Distress Syndrome (RDS) and/or BronchopulmonaryDysplasia (BPD). For treating RDS or BPD, the leptin compound comprisesat least a biologically active fragment of leptin which is administeredin a dosage from about 0.1 ng/kg body weight to about 100 mg/kg bodyweight. The leptin compound is administered by a method selected fromthe group consisting of subcutaneously, intradermally, intravenously,intramuscularly, intraperitoneally, transdermally, orally, enteral tubefeeding, pulmonary delivery, intranasal delivery, controlled releasedelivery and pump delivery.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a Western blot analysis of SP-B (surfactant protein-B) from21-day gestation, fetal rat lung explants cultured in the presence orabsence of leptin. Fetal lung explants were exposed to either (A) 1ng/ml or (B) 10 ng/ml leptin from initiation of culture (day 0).Explants were harvested and protein extracts were prepared on days (d)1, 2, and 3 of culture. Western blots were probed with an antibodyspecific to rat SP-B.

FIG. 2 shows RT-PCR analysis of surfactant protein A (SP-A), surfactantprotein B (SP-B), surfactant protein C(SP-C), and β-actin mRNAexpression in day 17 fetal lung explant cultures. Cultures were exposedto 1 ng/ml leptin (lanes 3, A-C) or control medium (lanes 2, A-C), andtotal RNA was isolated at the indicated day of culture. Total RNA wasreverse transcribed and amplified with both β-actin and leptin PCRprimers. In each figure, lane 1 is the time of initiation of culture(day 0). Low levels of all surfactant RNAs were detected in theuncultured cells (lanes 1, A-C). The size of the β-actin RT/PCR productis 492 bp; the size of SP-A is 352 bp; SP-B is 201 bp, and SP-C is 284bp.

FIG. 3 shows RT-PCR results of an experiment that determines therelative levels of the surfactant mRNAs to that of 18S rRNA in day 17fetal lung explants. Qualitative RT/PCR detection of surfactant mRNA wasperformed using the Ambion QuantumRNA kit (Ambion, Austin, Tex.) and 1μg of total RNA isolated from explants cultured for 3 days in culture.Lanes 1, 3, 5: control day 3; lanes 2, 4, 6: 1 ng/ml leptin day 3.

FIG. 4 depicts RT-PCR analysis of leptin receptor expression in day 17fetal lung explant cultures. Total RNA was reverse transcribed andamplified with either the short (OB-Ra) or the long (OB-Rb) form of theleptin receptor. Day 0 (Cd0) represents the time of initiation ofculture. The fetal lung explants were exposed for 3 days to eithercontrol medium (Cd3), 1 ng/ml leptin (Ld3), or 10 nM dexamethasone(Dexd3). The size of the OB-Ra RT/PCR product is 479 bp and OB-Rb is 262bp.

FIG. 5 depicts RT-PCR analysis of surfactant and GADPH (glyceraldehydephosphate dehydrogenase) mRNA expression in cultured isolated type IIalveolar cells. Cells were exposed to either 1 ng/ml leptin (1 L) or 10ng/ml leptin (10 L). Control cells were not exposed to leptin.

FIG. 6 depicts the effect of antenatal treatment with leptin ordexamethasone on fetal lung morphology. Fetal lung sections were stainedwith a histochemical stain for alkaline phosphatase, which identifiestype II cells. Control, no leptin; dexamethasone (6 mg/ml for 48 h);leptin (0.25 mg/ml for 48 h).

DETAILED DESCRIPTION OF THE INVENTION

The present invention includes a method for treating RDS by treatmentwith leptin. The invention is based on studies that show that leptin isproduced by the placenta, that leptin levels in cord blood arecorrelated with newborn birth weights, that leptin is produced by themammary gland and is found in breast milk, that leptin levels are higherin female as compared to male newborns, and that leptin augmentssurfactant production in fetal lung explant cultures. The combination ofthis role in fetal lung development with the known facts that theincidence of RDS mortality is lower in female as opposed to male infantsand lower in breast-fed than in formula-fed infants points to animportant role of leptin in maturation and function of the lung andforms the basis of the method of this invention.

Recent studies on the hormone leptin have outlined its role in energyhomeostasis, regulating such diverse processes as satiety, fetal andneonatal growth, and immune function (reviewed in Campfield, Smith, andBurn, Science 280: 1383-7, 1998). Leptin, a 167 amino acid cytokinehormone produced by the obesity (ob) gene, was initially thought to beadipocyte-specific (Zhang et al., Nature 372: 425-32, 1994). However,Hassink et al. (Pediatrics 100: e1-e6, 7, 1997) have discovered highlevel expression of leptin mRNA and protein in human placenta, andspeculated that leptin was produced by the syncytiotrophoblasts. Thiswas subsequently confirmed by Masuzaki et al. (Nat. Med. 3:1029-33,1997). Additionally, leptin has been shown to be produced by gastricepithelium (Bado et al., J. Clin. Endo. Metab. 82: 1642-5, 1998) and themammary gland (Smith-Kirwin et al., J. Clin. Endo. Metab. 83: 1810-3,1998). Furthermore, under conditions of nutrient deprivation, leptin isalso produced in skeletal muscle and induces its own expression in thistissue (Wang et al., Nature 393: 684-8, 1998; Nat. Med 5: 895-9, 1999).

Leptin regulates appetite and metabolic activity in mice(Rohner-Jeanreanaud and Jeanreanaud, N. Engl. J. Med. 344: 324-5, 1996)by acting through the long form of the leptin receptor (OB-Rb) in thehypothalamus (Campfield, Smith, and Burn, Science 280: 1383-7, 1998).Recently, additional roles for leptin have been suggested. Leptin hasbeen demonstrated to have angiogenic activity in vivo and in vitro(Sierra-Honigmann et al., Science 281: 1683-6, 1988). These studiesshowed that leptin induces neovascularization in cornea from normal ratsbut not fa/fa Zucker rats, which lack a functional leptin receptor. Inleptin-deficient (ob/ob) mice, puberty and pregnancy cannot beestablished without leptin administration (Chehab, Lim, and Lu, NatureGen. 12: 318-20, 1996), indicating that leptin may have a role in sexualmaturation and development (Hassink et al., Pediatrics 98: 201-5, 1996).Other roles for leptin include a regulator of hematopoeisis (Cioffi etal., Nature Medicine 2: 585-9, 1996; Gainsford et al., Proc. Natl. Acad.Sci. USA 93: 14564-8, 1996), glucose metabolism (Kamohara et al., Nature389: 374-7, 1997), and proinflammatory immune responses (Loffreda etal., FASEB J. 12: 57-65, 1998; Lord et al., Nature 394: 897-901, 1998).Leptin-deficient ob/ob mice were also noted to have a specificrespiratory phenotype of alveolar hyperventilation and chronichypercapnia (Tankersley et al., J. Appl. Physiol. 81: 716-23, 1996).These phenotypic abnormalities were noted in age and weight-matchedob/ob mice before pronounced obesity. In a subsequent study, Tankersleyet al. (J. Appl. Physiol. 85: 2261-9, 1998) demonstrated that leptinadministration to ob/ob mice ameliorated the volume-dependent decreasein lung compliance in these animals. leptin has also recently been shownto prevent respiratory depression in ob/ob mice (O'Donnell et al., Am.J. Resp. Crit. Care Med. 159: 1477-84, 1999). Because these ob/ob micewere born to either wild type or ob/+ mothers, the fetuses were exposedto placental and possibly, maternal, leptin in utero. Thus, it was notpossible to ascertain whether leptin affects fetal lung development inutero.

The observation that leptin is synthesized and secreted by human(Hassink et al., Pediatrics 100: e1-e6, 1997), rat (Chien et al.,Biochem. Biophys. Commun. 237: 476-80, 1997) and mouse (Hoggard et al.,Proc Nat Acad. Sci. 94: 1073-8, 1997) placental tissue has importantimplications, since it suggests a novel role for leptin in fetal growthand development. Because leptin expression was observed in humanplacenta from near-term pregnancies and in mouse placenta from 14.5 daybut not from 12 day of gestation (Tomimatsui et al., Biochem. Biophys.Res. Comm. 240:213-5, 1997; Hoggard et al., Proc. Nat. Acad. Sci.94:1073-8, 1997), it is suggested that leptin regulates some aspect ofdevelopmental growth in the fetus during the second half of gestation.In the human, leptin has been detected in cord blood as early as 18weeks of gestation (Jaquet et al., J. Clin. Endo. Metab. 83:1243-6,1998). Leptin levels in cord blood increase with gestation (Jaquet etal., J. Clin. Endo. Metab. 83:1243-6, 1998) and show a good correlationwith the birth weight of the newborn (Hassink et al., Pediatrics100:e1-e6, 1997; Matsuda et al., J. Clin. Endo. Metab. 82:1642-4, 1997),further supporting the hypothesis that leptin regulates fetal growth.Premature infants are delivered before the late pregnancy rise in leptinoccurs (Masuzaki et al., Nat. Med. 3:1029-33, 1997) and have low cordblood leptin levels (Highman et al., Am. J. Obstet. Gynecol. 178:1010-5,1998).

Without intending to be bound by theory, it is believed that leptin isimportant for lung growth and/or maturation and that the lack of leptinexposure late in pregnancy when the type II alveolar cells are maturingand producing surfactant could contribute to the respiratory distresssuffered by many premature infants. Since leptin has been found inamniotic fluid, leptin may have a direct effect on type II alveolar cellmaturation and growth, thereby increasing surfactant production.

The inadequacy and/or absence of pulmonary surfactant production atbirth is one of the most serious and life threatening problems faced bythe premature infant. Surfactant production is a maturation-dependentprocess, and deficiency results in RDS, which is characterized byinability to expand the alveoli and sustain adequate ventilation.Currently, treatment of RDS involves administering prenatal steroids tothe mother in an attempt to increase surfactant production prior todelivery, administration of exogenous surfactant to the premature infantafter birth, and supportive treatment with artificial ventilation untilthe premature infant's lungs mature. RDS accounts for a substantialburden of morbidity and mortality in premature infants, as well assignificant emotional and financial burdens on the family. Althoughsteroids increase serum leptin levels, there is recent evidence thatglucocorticoids interfere with leptin's interaction with its receptor(Ur et al., Horm. Metab. Res. 28:4744-7, 1996; Zakrzewska et al.,Diabetes 46:717-9, 1997). Furthermore, glucocorticoids may contribute tothe development of central leptin resistance (Zakrzewska et al.,Diabetes 46:717-9, 1997). These effects of glucocorticoids maycontribute to the poor growth observed in premature infants treated withsteroids. Therefore, it is desirable to provide a method for treatingRDS that does not involve the use of steroids.

The method of the present invention, which consists of administration ofa leptin compound to a patient, avoids the use of steroids whileproviding effective treatment for premature infants who suffer fromconditions in which there is insufficient production of surfactant. Themethod is also effective for treatment of newborns, infants, children,and adults who suffer from any condition caused by insufficientsurfactant production as administration of the leptin compound isexpected to increase surfactant production and improve lung function.

More particularly, the present invention provides a method for restoringpulmonary function in a patient who suffers from a lung diseasecharacterized by insufficient surfactant production. Abnormalities ofsurfactant production have been described in obstructive lung diseases,such as asthma, bronchitis, chronic obstructive pulmonary disease, andfollowing lung transplantation (reviewed in Grise, Eur. Resp. J. 13(6):1455-76, 1999). Abnormal surfactant production has also been seen ininfectious and suppurative lung diseases, such as cystic fibrosis,pneumonia, and AIDS. Finally, insufficient surfactant also characterizesdiseases such as acute respiratory distress syndrome (ARDS), pulmonaryedema, interstitial lung diseases, pulmonary alveolar proteinosis,following cardiopulmonary bypass, and in smokers. The method comprisesadministering a leptin compound to the host for a time and in an amountsufficient to restore or enhance respiratory function. Typically, theleptin compound will be administered in a dosage from about 0.1 ng perkg body weight to about 100 mg per kg body weight, for example.Effective amounts are determined by such factors as the leptincomposition, the mode of administration, the weight and general healthof the patient, and the judgment of the prescribing physician, forexample. Considerations associated with such factors are well known bythose persons skilled in the art.

Further, the present invention, which has been discussed in the contextof human patients, is not limited to use in humans, but is alsoeffective in treating respiratory conditions caused by inadequatesurfactant production in other mammalian species

In the treatment of premature infants, the leptin compound is typicallyadministered to any premature infant at increased risk of developing RDSfrom the onset of birth to the time when the infant would have reachedfull gestational age. For example, an infant born 4 months prematurelyis typically treated with leptin for 4 months or until lung function isrestored. For individuals that develop respiratory distress after birth,the individual is treated with leptin for a period of time until lungfunction is restored, as ascertained by clinical measurements that areknown to those skilled in the art.

The leptin compound may be administered subcutaneously, intradermally,intravenously, intramuscularly, intraperitoneally, via enteral tubefeeding, via pulmonary delivery, via intranasal delivery, transdermally,orally, via controlled release, via pump, or by any other conventionalroute of administration for polypeptide drugs. Typically, the leptincompound will be administered continuously during the period ofadministration, i.e., being delivered at least once per day or viacontrolled release techniques, such as via transdermal patches or leptinin milk fat globules. Furthermore, leptin may be administeredantenatally to those mothers at increased risk of deliveringprematurely. Leptin may also be administered antenatally withglucocorticoids. The administration of leptin has been described in U.S.patent application Ser. No. 09/302,117 which is incorporated herein byreference in its entirety.

Leptin may be derived from any mammal. Preferably human leptin is usedfor the treatment of humans. A leptin compound may include, but is notlimited to, either the full active peptide or a biologically activefragment that is capable of binding to the leptin receptor and elicitinga biological effect such as increased surfactant production. Methods forpurification of leptin, production of the recombinant form, and activebiological fragments have been described in U.S. Pat. Nos. 5,552,524;5,552,523; 5,552,522; 5,521,283; 5,5908,830, incorporated herein byreference in their entireties.

In some embodiments, the invention provides compositions foradministration which comprises a solution of leptin dissolved orsuspended in an acceptable carrier, preferably an aqueous carrier. Avariety of aqueous carriers may be used, e.g., water, buffered water,0.8% saline, 0.3% glycine, hyaluronic acid and the like. Thesecompositions may be sterilized by conventional, well-known sterilizationtechniques, or may be filter-sterilized. The resulting aqueous solutionsmay be packaged for use as is, or lyophilized, the lyophilizedpreparation being combined with a sterile solution prior toadministration. The compositions may contain pharmaceutically acceptableauxiliary substances as required to approximate physiologicalconditions, such as pH adjustments and buffering agents, adjustingagents, wetting agents and the like, for example, sodium acetate, sodiumlactate, sodium chloride, potassium chloride, calcium chloride, sorbitanmonolaurate, triethanolamine oleate, etc.

The concentration of leptin in the pharmaceutical formulations can varywidely, i.e., from less than about 0.1% to as much as 20% to 50% or moreby weight, and will be selected primarily by fluid volumes, viscosities,etc., in accordance with a particular mode of administration selected. Avalue of about 2% is common for many formulations, for example.

For solid compositions, conventional nontoxic solid carriers may be usedwhich include for example, pharmaceutical grades of mannitol, lactose,starch, magnesium sterate, sodium saccharin, talcum, cellulose, glucose,sucrose, magnesium carbonate, and the like. For oral administration, apharmaceutically acceptable nontoxic composition is formed byincorporating any of the normally employed excipients, such as thosecarriers previously listed, and generally 10% to 95% of activeingredient, that is one or more leptin compounds of the invention, andmore preferably at a concentration of 25% to 75%.

For aerosol administration, leptin is preferably supplied in finelydivided form along with an aerosol surfactant and propellant. Typicalpercentages of leptin are 0.01% to 20% by weight preferably 1%-10%. Theaerosol surfactant must, of course be nontoxic, and preferably suitableto the propellant. Representative of such agents are the esters orpartial esters of fatty acids containing from 6 to 22 carbon atoms suchas caproic, octanoic, lauric, palmatic, stearic linoleic, linolenic,olesteric and oleic acids with an aliphatic polyhydric alcohol or itscyclic anhydride. Mixed esters, such as mixed or natural glycerides maybe employed. The aerosol surfactant may constitute 0.1% to 20% by weightof the composition, preferably 0.25-5%. The balance of the compositionis ordinarily propellant. A carrier can also be included, as desired, aswith, e.g., lecithin for intranasal delivery.

The leptin compositions of the invention can additionally be deliveredin a controlled release system encapsulated form, or an implant bytechniques well known in the art. The compositions of the invention canalso be delivered via a pump, such as a minipump, or by administrationof milk fat globules containing leptin as disclosed in co-pending U.S.patent application Ser. No. 09/302,117.

EXAMPLES

1. Effects of Leptin on Surfactant Production in the Fetal Lung ExplantModel

We have used a fetal lung explant model to mimic the conditions of thepremature lung. Lungs from d21 rat fetuses, term being 22 days, weredissected free of heart, trachea and bronchi and placed in ice-coldserum free Waymouth medium and cut into 1 mm³ pieces on a McIlwaintissue chopper. The lung explants were placed in tissue culture dishesthat were scratched (along each half) to facilitate attachment ofexplants. The excess medium was aspirated and fresh medium (2 ml for 60mm plate) was gently placed on the explants. The 17-21 d explants wereincubated in 95% O₂-5% CO₂, since incubation of these explants in 5% CO₂in air can cause compression of airways (Gross and Wilson, J. Appl.Physiol. 55: 1725-32, 1983). The petri dishes were placed on a tiltingplatform and allowed to rest for 90 min in a humidified atmosphere in aCO₂ incubator. Thereafter, the plates were tilted at 3-4 cycles perminute so that during each cycle, one half of the petri dish was exposedto gas phase and the other half was covered with the medium. Leptin ateither 1 or 10 ng/ml was added to the explant cultures on the day ofestablishment of the culture (day 0). The explants were cultured forvarying periods of time for up to 3 days, and protein extracts wereprepared to establish a time course for leptin effects on the fetallung. Surfactant proteins were separated by electrophoresis on 15%polyacrylamide-SDS gels, and transferred to nitrocellulose membranes byelectroblotting. The membranes were blocked with 2% gelatin (BioRad),treated overnight with anti-SP-B, and then with secondary antibody (goatanti-rabbit HRP-conjugated antibody). The blots were then reacted with achemiluminescent substrate solution (SuperSignalR, Pierce Chemical Co.)and exposed to X-ray films to detect proteins that are recognized by theprimary antibody. A set of known concentrations of protein was run inparallel to ascertain that the amount of sample protein is within thelinear range of density.

FIG. 1 shows that 1 ng/ml leptin increases SP-B production above that ofthe control cultures after either 48 or 72 h of leptin exposure. Ahigher concentration of leptin (10 ng/ml) increases SP-B productionafter 24 h, but decreases SP-B levels after 48 and 72 h.

To explore the effect of gestational age on surfactant production inresponse to leptin, we exposed younger lung explant cultures (d17) to 1ng/ml leptin. Total RNA was extracted by homogenizing the explants in 4Mguanidine thiocyanate, applying the lysate on a Qiagen RNeasy column(Qiagen, Chatsworth, Calif.), and recovering total RNA according to themanufacturer's instructions. RNA was quantified by measurement ofabsorbancy at 260 nm (A₂₆₀). The quality of RNA was assessed by theA₂₆₀/A₂₈₀ ratio and by separation on agarose gels. Total RNA (1 μg) wasbrought up to 10 μl in DEPC-treated water. The sample was heated to 75°C. for 3 min, placed on ice, and cDNA synthesis was performed by reversetranscription for 15 min at 42° C. in a 20 μl reaction containing 1×PCRbuffer II (Perkin-Elmer), 5 mM MgCl₂, 1.25 mM each dNTP, 1 U/μl RNasin(Promega), 12.5 μg/μl oligo (dT) 15, and 2.5 U/μl AMV reversetranscriptase (Promega Madison, Wis.). Subsequent amplification of thecDNA sequence was performed with 10 μl of the reverse transcriptionreaction in 1× Taq buffer, 5% DMSO, 25 pmol each primer (Table 1), and1.25 U Taq polymerase in a 50 μl reaction volume. TABLE 1 Sequence ofPCR primers used in the RT/PCR experiments Primer Gene Sequence RSPAFRat SP-A 5′CCTCTTCTTGACTGTTGTCGCTGG3′ RSPAR Rat SP-A5′GCTGAGGACTCCCATTGTTTGCAG3′ RSPBF Rat SP-B 5′GGAGCTAATGACCTGTGCCAAGAG3′RSPBR Rat SP-B 5′CTGGCCCTGGAAGTAGTCGATAAC3′ RSPBR2 Rat SP-B5′AAGTACTGTGTAACGCTCAGCCAG3′ RSPCF Rat SP-C 5′GATGGAGAGCCCACCGGATTACTC3′RSPCR Rat SP-C 5′GAACGATGCCAGTGGAGCCAATAG ROBRaF Rat OB-Ra5′AGTGAATGCTGTGCAGTCACTCAG3′ ROBRaR Rat OB-Ra5′CAAAGAGTGTCCGCTCTCTTTTGG3′ ROBRbF Rat OB-Rb5′GGATGAGTGTCAGAGTCAACCCTC3′ ROBRbR Rat OB-Rb5′CAGTTCCAAAAGCTCATCCAACCC3′ ACTF1 Rat β-actin5′TGTATGCCTCTGGTCGTACCAC3′ ACTR1 Rat β-actin 5′ACAGAGTACTTGCGCTCAGGAG3′GAPDHF Rat GAPDH 5′GGTCGGTGTCAACGGATTTG3′ GAPDHR Rat GAPDH5′GAGATGATGACCCTTTTGGC3′

For assessment of the relative levels of SP-A, SP-B and SP-Ctranscripts, a multiplex RT/PCR reaction with β-actin was used. Thetemperature profile for the PCR reactions consisted of a 2 min meltingstep at 95° C., then 30 cycles of 30 s at 94° C., 30 s at 55° C., and 60sec at 65° C., followed by a final extension step of 5 min at 72° C.RT-PCR products were separated by size on a 4% agarose gel and stainedwith ethidium bromide. Gel visualization and quantitative analysis ofrelative band intensities was performed using Eagle Eye II hardware andsoftware (Stratagene, La Jolla, Calif.).

FIG. 2 shows that leptin significantly increases SP-A and SP-C mRNAlevels. However, leptin also increases the levels of β-actin in fetallung explant cultures. This results in an underestimation of the actualincrease in surfactant mRNA levels. Therefore, we chose to use 18S rRNAto determine the relative levels of surfactant mRNA because of theinvariant expression of 18S rRNA across tissues and treatments. Since18S rRNA is much more abundant than any mRNA species, modified 18S rRNAprimers called competimers (Ambion) are used that cannot be extended byTaq polymerase. By adjusting the ratio of competimers to normal 18S rRNAprimers, the RT/PCR signal for 18S rRNA can be decreased to the level ofeven rare messages, as described by the manufacturer. FIG. 3 shows theresults of such an experiment for determining the relative levels of thesurfactant mRNAs to 18S rRNA. FIG. 3 shows that leptin increases themRNA levels for SP-A, SP-B, and SP-C relative to the levels of 18S rRNAby 1.6-, 5-, and 2-fold, respectively, in d17 lung explant culturesafter 3 days in culture. These experiments support the hypothesis thatleptin has an effect on the maturation of type II alveolar cells.

2. Changes in Leptin Receptor Gene Expression in Relation to LungMaturation

Both the long (OB-Rb) and short (OB-Ra) forms of the leptin receptor areexpressed in fetal lung explant cultures (FIG. 4). As the fetal lungcells mature in culture, the expression of OB-Ra mRNA increases, whereasOB-Rb mRNA levels decrease. Leptin administration similarly decreasesexpression of OB-Rb mRNA, whereas dexamethasone increases OB-Rb mRNAlevels in fetal lung explants (FIG. 4). Taken together, these datademonstrate the presence of OB-Ra and OB-Rb mRNA in rat fetal explantlung cultures. As type II alveolar cells mature in culture, mRNA levelsof OB-Ra increase and OB-Rb decrease; leptin administration furtherdecreases OB-Rb mRNA levels, whereas dexamethasone increases OB-Rb mRNAexpression.

3. Leptin Increases mRNA Levels of Surfactant Proteins in Isolated FetalAlveolar Type II Cells

To determine if the leptin effect on surfactant production was due to adirect effect of leptin on the type II alveolar cell, we determinedwhether the effects of leptin could be reproduced in isolated type IIalveolar cells in culture. Alveolar type II cells were obtained from thelungs of 19-d gestation fetal rats by the method described by Bhandariet al., (Pediatr. Res. 41:166-71, 1997). In brief, lungs of 19-daygestation fetal rats were removed, dissected free of connective tissueand nonparenchymal pulmonary tissue, and cultured as explants for 40-48h in serum free Waymouth MB 752/1 medium with penicillin andstreptomycin in humidified 95% O₂/5% CO₂ at 37° C. During this time,endothelial and blood cells do not survive, which is a crucial step inthe enrichment of primary cultures of Type II cells from fetal lung. Theexplant cells were then harvested and the cells dissociated using asolution of collagenase, trypsin and DNase. The mixed cell suspensionwas subjected to three differential adhesions to remove fibroblasts. Thenon-adherent suspension containing an enriched population of fetal typeII cells was plated at 2×10⁶ cells/35 mm dish in 2 ml of minimumessential medium containing penicillin (100 U/ml), kanamycin (100 μg/ml)and 2% fetal bovine serum. The cells were cultured for 20-22 h at 37° C.in 5% CO₂/room air (Bhandari et al., Pediatr. Res. 41:166-71, 1997).Cultures contain 90-95% type II cells of which >99% are viable asdetermined by exclusion of the vital dye, trypan blue. The usual yieldof type II cells from the lungs of the fetuses (10-16) per pregnant ratis approximately 10⁷ cells.

Type II cells from d19 fetal lungs were cultured for 24 h and thenexposed to either 1 or 10 ng/ml leptin for 24 h. Total RNA was isolatedfrom these cells and then examined for SP-A, SP-B, SP-C, and GAPDH(housekeeping gene) mRNA expression. FIG. 5 shows that a 24 h treatmentwith 1 ng/ml leptin increased SP-A, SP-B, and SP-C, but not GAPDH, mRNAlevels after 24 h. These data suggest that leptin acts directly on thetype II cell and that the effects of leptin on SP-A, SP-B, and SP-C mRNAlevels are most likely exerted at the transcriptional level.

Taken together, these data demonstrate that leptin administration bothin fetal lung explant cultures and isolated type II alveolar culturesresults in increases in SP-A, SP-B, and SP-C mRNA. Lung explants fromday 21 fetuses produced increased surfactant proteins with leptinadministration, possibly indicating an effect on maturation of type IIalveolar cells. Thus, leptin provides a means to increase surfactantproduction in immature lungs, ultimately resulting in an additionaltreatment modality for premature infants with RDS and for otherconditions characterized by insufficient surfactant production.

4. Antenatal Treatment of Pregnant Rats with Leptin

Leptin (1 mg/kg body weight) was administered to pregnant rats at d16 ofgestation and 24 h later by intraperitoneal injection. Dexamethasone at6 mg per kg body weight was administered at similar time intervals asleptin. In addition, the effect of a combination of leptin anddexamethasone treatment was tested. After 48 h of leptin, dexamethasoneor leptin/dexamethasone exposure, premature delivery of the rat pups wasinduced at d18, which is similar to 30 weeks of gestation in the human.The fetuses from each litter were pooled and weighed, and varioustissues from both the rat fetuses and mothers were also dissected andweighed. Table 2 shows that antenatal treatment with leptin increasedthe average weight of the fetal lungs in relation to their body weightby 51%. Antenatal treatment with dexamethasone increased fetal lungweight by 41%. Interestingly, combined therapy with leptin anddexamethasone increased fetal lung weight by 62%. TABLE 2 Effect ofantenatal treatment with dexamethasone, leptin, or dexamethasone andleptin on weight of maternal and fetal lungs Control DexamethasoneLeptin Dex + Leptin Maternal lung 1.07 0.99 1.21 1.16 weight (g) Averageweight 0.61 0.79 0.87 0.95 fetal lungs (g) (n = 9) (n = 9) (n = 9) (n =9) Average weight 0.47 0.43 0.45 0.47 fetal head (g) (n = 9) (n = 9) (n= 9) (n = 9) Average weight 1.64 1.52 1.56 1.59 fetus (g) (n = 9) (n =9) (n = 9) (n = 9) Weight fetal 0.37 0.52 0.56 0.60 lung (g)/g fetus

Histologic analysis of the lung tissue was done to determine the basisfor the increased fetal lung weight. Type II cells were identified byalkaline phosphatase staining as previously described (Post and Smith,Am. Rev. Respir. Dis. 137: 525-30, 1988). Unfixed frozen 5 μm sectionsof fetal lung tissue were stained histochemically with alkalinephosphatase at pH 8.74 to identify type II alveolar cells. Slides wereincubated for 60 minutes at 37° C. in 25 ml of 0.2M Tris-HCl buffer and25 ml of deionized water, 5 mg of Naphthol AS-BI phosphate, 0.1 mldimethylformamide, and 30 mg of fast red TR. The slides were then rinsedin 3 changes of deionized water, counterstained for 30 seconds in Harrishematoxylin and blued in running water for 3-5 minutes. Slides weremounted from water in Advantage™. The histologic analysis revealed thatthe increase in fetal lung weight in both the leptin- anddexamethasone-treated rats was paralleled by an increase in the numberof type II alveolar cells (FIG. 6).

It will be readily understood by those persons skilled in the art thatthe present invention is susceptible to broad utility and application.Many embodiments and adaptations of the present invention other thanthose herein described, as well as many variations, modifications andequivalent arrangements, will be apparent from or reasonably suggestedby the present invention and foregoing description thereof, withoutdeparting from the substance or scope of the invention.

Accordingly, while the present invention has been described here indetail in relation to its preferred embodiment, it is to be understoodthat this disclosure is only illustrative and exemplary of the presentinvention and is made merely for the purposes of providing full andenabling disclosure of the invention. The foregoing disclosure is notintended to be construed or to limit the present invention or otherwiseto exclude any other such embodiments, adaptations, variations,modifications and equivalent arrangements, the present invention beinglimited by the claims and the equivalents thereof.

1-16. (canceled)
 17. A method for regulating appetite activity in asubject comprising administering an effective dose of a non-recombinantbiologically active fragment of leptin to the subject.